ES2494817T3 - New mite allergen - Google Patents

Abstract

The following recombinant mite allergen (a) to (c) having mite allergenic activity: (a) a recombinant mite allergen comprising the amino acid sequence represented by SEQ ID NO: 35; (b) a recombinant mite allergen comprising the amino acid sequence of SEQ ID NO: 35, except that 1 to 10 amino acids have been deleted from the amino acid sequence represented by SEQ ID NO: 35; or (c) a recombinant mite allergen comprising an amino acid sequence that has at least 85% sequence homology to the amino acid sequence of SEQ ID NO: 35 calculated using BLAST and using default parameters.

Description

New mite allergen

Technical field

The present invention relates to recombinant mite allergens that have allergenic activity and in particular refers to mite allergens that cause atopy in dogs. The present invention further relates to genes encoding allergens, expression vectors that enable gene expression, transformants obtained by transformation using expression vectors, a method for producing recombinant mite allergens, therapeutic agents for mite allergic diseases , and diagnostic agents for allergic diseases to mites.

Background Technique

House dust mites are known as major causes of allergic diseases such as atopic dermatitis and bronchial asthma. Conventionally, desensitization therapy using allergy-causing substances as therapeutic agents for allergic diseases is considered as the most important basic remedy. In particular, desensitization therapy is widely performed for diseases such as polynose, household dust allergy, and fungal allergies, which are induced by antigens such as inhalant allergens that are difficult to avoid. However, desensitization therapy involves the risk of anaphylaxis due to the action of sensitizing antigens, so that the administration of safe therapeutic antigens is required. Such safe sensitizing antigens are under investigation.

Regarding mite allergic diseases, types of mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae, have been reported as allergenic sources in household dust (see non-patent documents 1 and 2). The main mite allergens have been fractionated from these mites. It is known that these mite allergens are a glycoprotein (pI 4.6 to 7.2) with a molecular weight between 24 kD and 28 kD and / or a protein (pI 5 to 7.2) with a molecular weight between 14, 5 kD and 20 kD contained in the excretion of mites and / or mite bodies (see non-patent documents 3 to 7).

Regarding the mite allergen genes, Der p 1 (molecular weight: 25,371) and Der p 2 (molecular weight: 14,131) that are major allergens of Dermatophagoides pteronyssinus and Der f 1 (molecular weight: 25,191) and Der f2 (weight molecular: 14,021) which are major allergens of Dermatophagoides farinae have been cloned and nucleotide sequences thereof have also been determined (see non-patent documents 8 to 15). Recombinant allergens based on these allergens have also been prepared, and research regarding them has progressed. In addition, the nucleotide sequence of Der f 3, an allergen with a molecular weight of approximately 30,000, has also been presented (see non-patent document 16). In addition, as mite allergens, ma 10, ma 3, ma 15, ma 29, ma 44, ma 50, ma 113, ma 114, and ma 115 (see patent document 1) have also been submitted. In addition, ma 124, which exerts strong cross reactivity with an anti-Der f2 serum, has also been presented (see patent document 2).

In addition, it has been reported with respect to dogs that Der f 15 of 98 kDa, Der f 15 of 109 kDa (see non-patent document 17), and Der f 18 of 60 kDa (see non-patent document 18) are allergens with which IgE reacts strongly.

As a method to diagnose mite allergic diseases, an intradermal reaction assay has been conventionally used as the dominant method, which is based on the patient's history and uses household dust extracts and / or mite body extracts. This method (test) has been used in combination with a RAST method (allergy-absorbing radio test) that involves the measurement of serum IgE antibody titre (relative value), an inhalation induction test, a nasal mucous membrane provocation test , and the like. However, it has remained considerably difficult to directly diagnose mite allergic diseases.

A therapeutic method of desensitization for bronchial asthma, which uses a household dust extract and a house dust mite as a specific allergen, has been conventionally performed. However, the composition of household dust has not been sufficiently analyzed. In addition, household dust contains many types of impurities that can induce anaphylaxis. Therefore, the doses of household dust in such cases are extremely limited. Accordingly, conventional desensitization treatment can be affected at extremely low levels. Therefore, more effective and safer antigens have been desired for desensitization treatment. It has been conventionally known that allergens effective for desensitization treatment are present in high molecular weight raw mite excretion fractions. From these fractions, it has been impossible to obtain mite allergens in amounts sufficient for desensitization treatment. Therefore, with methods that involve the extraction and purification of mite allergens from products obtained by mite breeding, it is difficult to achieve a stable supply of antigens for treatment. In addition, as described above, various mite recombinant allergens have been conventionally reported with the use of gene recombination techniques. However, it cannot be said that these

Allergens are always effective for the real treatment. The provision of a recombinant mite allergen with more effective, new, and higher activity of mite allergen has been desired.

Patent document 1 patent publication JP (Kokai) No. 7-112999 A (1995) Patent document 2 patent publication JP (Kokai) No. 7-278190 A (1995) Non-patent document 1 Allerg. Asthma, 10, 329-334 (1964) Non-patent document 2 J. Allergy, 42, 14-28 (1968) Non-patent document 3 J. Immunol., 125, 587-592 (1980) Non-patent document 4 J. Allergy Clin. Immunol., 76, 753-761 (1985) Non-patent document 5 Immunol., 46, 679-687 (1982) Non-patent document 6 Int. Arch. Allergy Appl. Immunol., 81, 214-223 (1986) Non-patent document 7 J. Allergy Clin. Immunol., 75, 686-692 (1985) Non-patent document 8 Int. Arch. Allergy Appl. Immunol., 85,127-129 (1988) Non-patent document 9 J. Exp. Med., 167, 175-182 (1988) Non-patent document 10 J. Exp. Med., 170, 1457-1462 (1989) Non-patent document 11 Int. Arch. Allergy Appl. Immunol., 91, 118-123 (1990) Non-patent document 12 Int. Arch. Allergy Appl. Immunol., 91, 124-129 (1990) Non-patent document 13 Jpn. J. Allergol., 39, 557-561 (1990) Non-patent document 14 Clinical and Experimental Allergy, 21, 25-32 (1991) Non-patent document 15 Clinical and Experimental Allergy, 21, 33-37 (1991) Non-patent document 16 FEBS Lett., 377, 62-66 (1995) Non-patent document 17 Vet. Immunol Immunopathol, 78, 231-247 (2001) Non-patent document 18 J. Allergy Clin. Immunol, 112, 79-86 (2003)

Description of the invention

An object of the present invention is to provide effective mite allergens that do not contain impurities that induce anaphylaxis as therapeutic agents or diagnostic agents for mite allergic diseases. More specifically, the objectives of the present invention are to provide genes derived from mite bodies and to provide expression vectors that enable gene expression. A further objective of the present invention is to provide new mite allergens that have allergenic activity, which are obtained by expression of genes derived from mite bodies. Additional objectives of the present invention are to provide new therapeutic agents for mite allergic diseases that contain recombinant mite allergens as active ingredients and to provide new diagnostic agents for mite allergic diseases that contain recombinant mite allergens.

As a result of intensive studies to achieve the above objectives, the present inventors have discovered new mite allergens and have also discovered that the allergens exert excellent effects in desensitization treatment. Therefore, the present inventors have completed the present invention.

Specifically, the present invention is as described below.

[1] The following mite (a) to (c) recombinant allergen that has mite allergenic activity:

(to)

a recombinant mite allergen comprising the amino acid sequence represented by SEQ ID NO: 35;

(b)

a recombinant mite allergen comprising the amino acid sequence of SEQ ID No. 35, except that they are deleted from 1 to 10 amino acids of the amino acid sequence represented by SEQ ID No. 35; or

(C)

a recombinant mite allergen comprising an amino acid sequence that has at least 85% sequence homology with the amino acid sequence of SEQ ID No. 35 calculated using BLAST using default parameters.

[2] A gene that (1) encodes the following mite allergen (a) to (c) that has mite allergenic activity:

(to)

a mite allergen comprising the amino acid sequence represented by SEQ ID NO: 35;

(b)

a mite allergen comprising the amino acid sequence of SEQ ID No. 35, except that they are deleted from 1 to 10 amino acids of the amino acid sequence represented by SEQ ID No. 35; or

(C)

a recombinant mite allergen comprising an amino acid sequence that has at least 85% sequence homology with the amino acid sequence of SEQ ID No. 35 calculated using BLAST using default parameters: or

(2)

It comprises the following DNA (d) or (e):

(d)

a DNA comprising the nucleotide sequence represented by SEQ ID NO: 34; or

(and)

a DNA encoding a protein that has mite and hybrid allergen activity under stringent conditions with a DNA comprising a sequence complementary to that of the DNA comprising the

nucleotide sequence of SEQ ID No. 34, where the DNA (1) hybridizes in the presence of 0.7 M NaCl at 1.0 M at 68 ° C using a filter in which the DNA is immobilized and washed with the use of SSC solution 0.1 to 2 x 68 ° C, or

(2) It may form a hybrid when transferred to and immobilized on a nitrocellulose membrane by the Southern transfer method and then allowed to react overnight at 42 ° C in a 50% formamide hybridization buffer, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's solution and 10 µg / ml salmon sperm DNA.

[3] The mite allergen according to [1] part (c) or the gene according to [2] part (c), where the homology level is at least 90%.

[4] The mite allergen or gene that encodes [3], where the homology level is at least 95%.

[5] The mite allergen or gene according to [3], where the homology level is at least 97%.

[6] A recombinant vector, which contains the gene according to any one of [2] to [5].

[7] A fusion protein, which is composed of the mite allergen according to any one of [1] and

[3] to [5] and another protein.

[8] A bacterial, yeast, insect, or animal cell, which is transformed using the expression vector according to [6].

[9] A method for producing a recombinant mite allergen, comprising culturing the bacterial, yeast, insect or animal cell according to [8] under conditions where the gene can be expressed, causing the cell to produce a recombinant allergen of mite, and then collect the recombinant mite allergen.

[10] A method of producing a recombinant mite allergen, comprising culturing a bacterial, yeast, insect or animal cell that is transformed with a recombinant vector containing a gene that encodes a fusion protein between the mite allergen of any one of [2] - [5] and another protein under conditions in which the gene can be expressed, cause the cell to produce a recombinant fusion mite allergen, collect the recombinant fusion mite allergen, and then remove the other protein fused to the allergen.

[11] A therapeutic agent for mite allergic diseases, which contains as an active principle the recombinant mite allergen according to any one of [1] and [3] to [5], or the fusion protein according to [7 ].

[12] A diagnostic agent for mite allergic diseases, which contains as an active principle the recombinant mite allergen according to any one of [1] and [3] to [5], or the fusion protein according to [ 7].

[13] An antibody against the mite allergen according to any one of [1] and [3] to [5].

[14] The antibody against the mite allergen according to [13], which is a monoclonal antibody.

[15] A hybridoma, which produces the monoclonal antibody according to [14].

[16] An immunoassay method for a house dust mite allergen, which uses the antibody according to any one of [13] to [15].

[17] The immunoassay method for a house dust mite allergen according to [16], which is the ELISA method.

Brief description of the drawings

Fig. 1 is a photograph showing the electrophoresis result of a dog FcεRIα chain recombinant Fig. 2 is a graph showing the binding of a recombinant dog FcεRIα chain with IgE. Fig. 3 shows the result of detecting specific IgE of Dermatophagoides farinae. Fig. 4 is a photograph showing the results of Western blot analysis using a FcεRIα chain of recombinant dog. Fig. 5 is a photograph showing a 2-D (two-dimensional) electrophoresis pattern of an extracted antigen from Dermatophagoides farinae. Fig. 6 shows photographs showing the results of 2-D electrophoresis (two-dimensional) and analysis of Western transfer of an allergenic protein from Dermatophagoides farinae. Fig. 7-1 shows the partial nucleotide sequence and amino acid sequence of a Zen1 gene. Fig. 7-2 shows the partial nucleotide sequence and amino acid sequence of the Zen1 gene (continued from Fig. 7-1). Fig. 8-1 shows the full length cDNA nucleotide sequence and amino acid sequence of the Zen1 gene. Fig. 8-2 shows the full length cDNA nucleotide sequence and amino acid sequence of the Zen1 gene (continued from Fig. 8-1). Fig. 8-3 shows the full length cDNA nucleotide sequence and amino acid sequence of the Zen1 gene (continued from Fig. 8-2). Fig. 8-4 shows the full length cDNA nucleotide sequence and amino acid sequence of the Zen1 gene (continued from Fig. 8-3). Fig. 9 is a photograph showing the result of SDS-PAGE of recombinant Zen1 prepared using Escherichia coli and then purified. Fig. 10 is a photograph showing the result of a reaction analyzed by Western blotting of a polyclonal anti-Zen1 antibody. Lane 1 shows the result with respect to recombinant Zen1 and Lane 2

It shows the result with respect to a mite body. Fig. 11 is a graph showing the reactivity of recombinant Zen1 with IgE analyzed by ELISA.

Best way to carry out the invention

5 The present invention will be described in detail as follows.

(1) Isolation of a mite allergenic Zen1 protein and determination of partial sequences thereof

10 A mite allergen is specified using specific mite allergen IgE obtained from a clinically diagnosed animal as having a mite allergy. Specifically, a mite allergen is identified by Western blotting using serum containing mite allergen specific IgE, a mite extract, and an IgE receptor that recognizes allergen specific IgE, for example. An allergen can be identified by a known method.

The new mite allergen identified in this way of the present invention, which is a Zen1 protein, has a molecular weight between 150 kDa and 200 kDa.

The identified mite allergen can be isolated by electrophoresis and then by removing the mite allergen 20 from a mite allergen spot. At that time, it is desired to perform 2-D electrophoresis (two-dimensional) for the complete separation of other proteins.

Partial sequences can be determined by a known method using the mite allergen extracted in this way. Examples of known methods for determining partial sequences include the de novo sequencing based on MS / MS and peptide mapping.

(2) Preparation of cDNA clone by RT-PCR

A DNA encoding the mite allergen of the present invention can be obtained by extracting mRNA from a mite, synthesizing a mite allergen cDNA using the mRNA as a template, building a cDNA library, and then selecting the target.

A source of supply of said mRNA is a mite body, and the mite is preferably Dermatophagoides farinae, Dermatophagoides pteronyssinus, or the like, which are house dust mites. However, the 35 examples are not limited to them. Said mRNA can be prepared by generally employed techniques. The mRNA thus obtained is used as a template, primers are designed based on the sequence information obtained in (1) above, and then a cDNA fragment encoding a mite allergen is synthesized. The fragment obtained in this way is sub-cloned into an appropriate vector such as pGEM (produced by Promega). The nucleotide sequence is then determined by a conventional method such as a method of

40 cyclic sequencing.

The partial amino acid sequences of the mite allergen of the present invention are shown in SEQ ID NOS 3 to 19. Of these, the sequences shown in SEQ ID NOS 3 to 7 were determined by de novo sequencing. An N-terminal amino acid sequence is shown in SEQ ID No. 19. The sequences shown in SEQ ID.

No. 8 to 18 were determined by peptide mapping.

This document describes a mite allergen comprising an amino acid sequence comprising at least one of the amino acid sequences shown in SEQ ID NOS 3 to 19 that represent fragments of the Zen1 protein, which is a mite allergen.

A partial nucleotide sequence of the cDNA of the Zen1 gene encoding the Zen1 protein is shown in SEQ ID No. 1, and the full length nucleotide sequence thereof is shown in SEQ ID No. 34. A partial amino acid sequence. of the Zen1 protein is shown in SEQ ID No. 2, and the full length amino acid sequence thereof is shown in SEQ ID No. 35.

55 Whenever a protein comprising said amino acid sequence has mite allergenic activity, mutations such as deletion, substitution, or addition of at least one, and preferably one or more, amino acids in the amino acid sequence can take place.

For example, at least one, and preferably one or more (for example, 1 to 10 and more preferably 1 to 5), amino acids of the amino acid sequence represented by SEQ ID NO. 2 or SEQ ID NO.

35. At least one, and preferably one or more (for example, 1 to 10 and more preferably 1 to 5), amino acids can be added to the amino acid sequence represented by SEQ ID NO. 2. Alternatively, at least one can be substituted , and preferably one or more (for example, 1 to 10 and more preferably 1 to 5), amino acids of the

65 amino acid sequence represented by SEQ ID No. 2 with another or other amino acids.

Examples of said amino acid sequence derived from the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 35 by deletion, substitution, or addition of one or more amino acids include amino acid sequences having at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more homology with the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 35 calculated using BLAST (Local Alignment Search Tool Basic of the National Center for Biological Information) using default parameters for initial configuration, for example.

A protein having said amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2

or SEQ ID No. 35 by deletion, substitution, or addition of one or more amino acids is substantially equal to the protein having the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 35.

In addition, examples of the gene of the present invention also include a DNA that is capable of hybridizing under the following conditions with a DNA comprising a sequence complementary to that of a gene having the DNA sequence shown in the above SEQ ID NO: 34 and which encodes a protein that has mite allergenic activity. Specifically, such conditions allow identification by hybridization in the presence of 0.7 M NaCl at 1.0 M at 68 ° C using a filter in which a DNA is immobilized and washing with the use of a 0.1 to 2 X SSC solution ( 1 X SSC comprises 150 mM NaCl and 15 mM sodium citrate) at 68 ° C. Alternatively, the gene of the present invention is a DNA that can form a hybrid when transferred to and immobilized on a nitrocellulose membrane by the Southern transfer method and then allowed to react overnight at 42 ° C in a hybridization buffer ( 50% formamide, 4 X SSC, 50 mM HEPES (pH 7.0), 10 X Denhardt solution, and 100 µg / ml salmon sperm DNA).

Also described is an RNA that corresponds to the previous DNA or an RNA capable of hybridizing under stringent conditions with the RNA and encoding a protein that has mite allergenic activity.

The recombinant vector of the present invention is as set forth in the claims and can be obtained by linking (inserting) the gene of the present invention into an appropriate vector. Vectors for use in the insertion of the gene of the present invention are not particularly limited as long as they can be replicated in hosts such as bacterial, yeast or animal cells. Examples of such vectors include a plasmid DNA and a phageal DNA. A vector DNA that is used for the construction of an expression vector is widely disseminated and easily obtained. Examples of said vector DNA include pUC19 and pTV118 N (produced by Takana Shuzo), pUEX2 (produced by Amersham), pGEX-4T and pKK233-2 (produced by Pharmacia), and pMAM-neo (produced by Clontech).

A method for constructing said expression vector of the present invention is not particularly limited and can be performed according to a conventional method. For example, an mite allergen cDNA fragment digested with EcoR I can be inserted into the EcoR I site at the multiple cloning site of plasmid pUC19. In addition, the fragment can be ligated to the EcoR I site of the plasmid vector pGEX-4T, so that an expression vector can be obtained.

Bacterial, yeast or animal cells transformed with said expression vector of the present invention are not particularly limited, provided they can express the gene of the present invention. Examples of such bacteria include Escherichia coli and Bacillus subtilis. Examples of such yeasts include Saccharomyces cerevisiae and the like. Examples of such animal cells include Chinese hamster ovary (CHO) cells, Sf21 and Sf9 cells that are Mamestra brassicae ovary cells, monkey COS cells, and mouse fibroblasts.

Examples of the mite recombinant allergen of the present invention include, in addition to the mite allergens that are directly expressed, those expressed as fusion proteins with other proteins as set forth in the claims. As of now in this document, said fusion proteins are mentioned as recombinant fusion mite allergens. Examples of other proteins that form said fusion proteins include, but are not limited to, β-galactosidase, glutathione S-transferase, protein A, and a maltose binding protein.

Also described herein is a peptide fragment consisting solely of a region essential for allergenic activity or a peptide fragment comprising a region essential for allergenic activity. In addition, apart from a product obtained by expression of an mite allergenic protein only, said recombinant mite allergen can be obtained from a product expressed as a fusion protein by eliminating the other or other proteins.

Specifically, the mite recombinant allergen of the present invention is obtained by expression of a gene derived from a mite body and is a protein that has mite allergenic activity. Here, "which has mite allergenic activity" means that it is capable of inducing an allergic reaction in a mammal.

The mite allergen of the present invention can be produced by the following methods. After the culture of the preceding transforming strain is completed, the microbial bodies are collected, suspended in a

buffer containing various protease inhibitors, and then altered by ultrasonication. A membrane localized protein is extracted into cell debris using a buffer containing a protease inhibitor such as phenylmethanesulfonyl fluoride, monoiodoacetic acid, pepstatin A, or ethyllenediaminetetraacetic acid and a surfactant such as sodium lauryl sulfate (SDS), triton X- 100, or Nonidet P40. A fusion protein composed of the mite and glutathione S-transferase allergen obtained from the extract or the culture concentrate is purified by affinity chromatography using immobilized glutathione, affinity chromatography using immobilized mite anti-body antibody, or the like. In addition, a vehicle in which glutathione has been immobilized is a vehicle produced by Pharmacia. In addition, a vehicle in which an anti-mite body antibody has been immobilized is a vehicle prepared by covalently binding an anti-body mite rabbit antibody against an activated Tresyl vehicle (e.g., Tresil GM gel (produced by Kurita Water Industries), Tresil Toyopearl (produced by Tosoh), and Tresil sepharose (produced by Pharmacia)). In addition, a fusion protein composed of mite allergen and a His brand (for example, 6X His) can be obtained, followed by purification using affinity beads in which a metal, or the like, has been immobilized.

The recombinant purified fusion mite allergen is digested with protease and then fractionated by a single method or a combination of known purification methods including gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromato-focus , an isoelectric focusing method, and a gel electrophoresis method while a control with ELISA and a leukocyte histamine release assay for patients with mite allergic disease takes place (Allergy 37, 725 (1988)).

The present invention also encompasses a therapeutic agent for mite allergic diseases that contains an mite allergen as an active ingredient as set forth in the claims. Said therapeutic agent is used as a therapeutic agent for various types of mite allergic diseases. Here, "allergic diseases to mites" means all allergic diseases that are caused by specific mite antigens, such as atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis.

The therapeutic agent for mite allergic diseases of the present invention can be prepared by drying a recombinant mite allergen or a peptide fragment thereof purified by the above method, collecting said allergen or peptide fragment in a powder form, and then preparing a therapeutic agent. desensitizer for allergic diseases to mites, for example. However, the method is not particularly limited to it. When the therapeutic agent for mite allergic diseases of the present invention is used as a desensitizing therapeutic agent, the agent can be used directly or, if necessary, used as a combination drug supplemented by a conventional method with a generally used adjuvant and various additive agents. such as a stabilizing agent, an excipient, a solubilizing agent, an emulsifying agent, a buffering agent, a calming agent, a preservative, and a coloring agent. For example, a purified mite allergen is dissolved in a powdered form in physiological saline supplemented with phenol and then used as the stock solution for an antigen for desensitization treatment.

The therapeutic agent for mite allergic diseases of the present invention can be administered by general routes of administration such as percutaneous, oral, intracutaneous, subcutaneous, intramuscular, and intraperitoneal administration methods. In addition, the therapeutic agent of the present invention can also be used in a percutaneous or transmucosal drug such as a trocisco, a sublingual tablet, a eye drops, an intranasal spray agent, a poultice, a cream, and a lotion. In addition, the dose and the number of times of administration of the therapeutic agent for mite allergic diseases of the present invention are appropriately selected depending on the route of administration, the symptoms, and the like, so that the dose is within a range of approximately 20 µg or less per time of administration for an adult. Administration is done once or several times a week.

In addition, the therapeutic agent for mite allergic diseases of the present invention is useful not only as a therapeutic agent against mite allergic diseases, but also as a prophylactic agent against them. In addition, the therapeutic agent for mite allergic diseases of the present invention can be used safely in human organisms without exerting anaphylaxis inducing action.

The diagnostic agent for mite allergic diseases of the present invention is as set forth in the claims and is used as a reagent to diagnose intracutaneous reactions against mite allergic diseases or a titration reagent to diagnose mite allergies. When the diagnostic agent is used as a reagent to diagnose intracutaneous reactions, the reagent is obtained by preparing a recombinant mite allergen or a peptide fragment thereof purified by the above method according to a conventional method. For example, a recombinant mite allergen is dried and sprayed, the powder is dissolved and diluted in physiological saline containing phenol, and then used. A method is used that uses the diagnostic agent as a reagent to diagnose intracutaneous reactions according to a conventional method.

In addition, when the diagnostic agent is used as a titration reagent to diagnose mite allergies, the reagent is similarly prepared by a conventional method. For example, an appropriately dissolved

Recombinant mite allergen or a peptide fragment thereof and diluted in a Hank buffer, so that the resultant is used as a reagent for histamine release titration. This method is generally performed by the following procedures. Specifically, blood is suspended from a patient with mite allergic disease or a fraction of the blood cells obtained by centrifuging the patient's blood in a buffer. A fixed amount of the blood cell suspension is subjected to titration using a recombinant mite allergen as a titration reagent. The amount of histamine released from vasophiles is measured by allergenic stimulation using HPLC [Allergy 37, 725 (1988)].

In the histamine release titration, the amount of histamine that is released is determined based on 50% (inflection point of the titration curve) of the maximum release amount. Specifically, this titration is characterized in that: (1) a patient's allergen sensitivity is measured directly based on a titer of a blood cell suspension; and (2) after pre-reaction of the blood plasma with a recombinant mite allergen, the value (value of the blood titration curve) obtained by titration of the blood cell suspension with the reaction solution is usually greater than the value (value of the titration curve of the blood cell suspension) obtained by titrating the blood cell suspension with the recombinant mite allergen. This is due to the presence of an IgG antibody (blocking antibody) capable of neutralizing the allergen in blood plasma. Therefore, the titer of the blocking antibody can be obtained from the degree to which the blood titration curve has shifted from the blood cell suspension titration curve. Allergen sensitivity and this blocking antibody titer enable a precise feasible mite allergy diagnosis. This histamine release titration assay is also useful for controlling the effect of desensitization treatment.

The present invention also encompasses an antibody against the mite allergen of the present invention or a peptide fragment thereof as set forth in the claims. Said antibody can be obtained as a polyclonal antibody or monoclonal antibody by a known method. Said antibody can be used to measure the presence, absence, or the like of a house dust mite allergen, for example. Said measurement can be performed by a known immune method such as ELISA. After such measurement, a protein is extracted from the household powder and then measured.

In addition, the mite recombinant allergenic protein of the present invention is expressed. By performing an assay such as a specific IgE reaction assay or an intracutaneous reaction assay with a patient mite allergy dog using the recombinant protein expressed in this way, the allergen protein functions of the mite allergenic protein of the mite can be confirmed. present invention

The present invention will be further described in the following examples. The examples are not intended to limit the scope of the invention.

In addition, the reagents used in each example were commercial reagents purchased from Nacalai Tesque, Wako Pure Chemical Industries, Sigma, Difco, or the like, unless otherwise specified. In addition, reagents for genetic engineering, such as restriction enzymes, were purchased from Takara Shuzo, Toyobo, Invitrogen, or the like and then used according to the manufacturer's instructions.

[Example 1] Establishment of an IgE detection system

The extracellular region of a high-affinity affinity dog IgE receptor (FcεRIα) α-cDNA was amplified, excluding its signal peptide site and having EcoRI and Xho I restriction enzyme sites added thereto, by PCR. The resultant was ligated into the EcoRI and Xho I sites of the Escherichia coli pGEX4T-1 plasmid expression vector (produced by Amersham Biosciences) using T4 DNA ligase. A strain of E. coli TOP10 (produced by Invitrogen) was transformed with the recombinant plasmid thus obtained. The transformed strain was grown at 37 ° C overnight in an LB medium containing ampicillin (100 µg / ml). Subsequently, a small amount of the strain was subculitted in a new LB medium until the OD at 600 nm reached 1.0. Next, IPTG (isopropyl-1-thio-β-D-galactoside) was added to a final concentration of 1 mM. Three hours later, the cells were collected and then washed once with PBS (pH 7.4). The governed cells were again lysed by ultrasonication in PBS (pH 7.4), the insoluble fractions were removed by centrifugation, and then the soluble fractions containing FcεRIα fused to glutathione S-transferase (GST) were collected. Subsequently, FcεRIα from fused dog a of the soluble fractions was obtained using a sepharose 4B glutathione column (produced by Amersham Biosciences). 1.0 mg of the fusion protein (GST-FcεRIα) of 10 liters of the culture solution was obtained. It was confirmed that the purified GST-FcεRIα obtained showed a single band of 45 kDa as a result of SDS-PAGE (Fig. 1). Recombinant dog IgE (produced by BETHYL) and purified dog IgG were immobilized in an immunoplate (produced by Nalge Nunc International) with serial dilution of factor 2 of 1.0 µg, 0.1 µg, 0.05 µg , 0.025 µg, 0.0125 µg, and then 0.00625 µg, to confirm the reactivity of the purified GST-FcεRIα. It was confirmed that the purified GST-FcεRIα reacted with IgE but did not react with IgG (Fig. 2). 4.0 µg of an antigen extracted from Dermatophagoides farinae (produced by GREER) was immobilized in an immunoplate (produced by Nalge Nunc International). Dermatophagoides-farinae-positive dog serum was reacted (dog serum that tested positive for

Dermatophagoides farinae confirmed by an intracutaneous reaction using an antigenic solution of Dermatophagoides farinae (produced by GREER) diluted 50 times using physiological saline) with the antigen. Then GST-FcεRIα labeled with biotin was added. In addition, based on the color development reaction resulting from the addition of peroxidase-conjugated streptavidin (produced by Jackson Immuno Research) and a substrate, it was confirmed that GST-chain FcεRIα recognized mite allergen-specific IgE (Fig. 2). In addition, 4.0 µg of an antigen extracted from Dermatophagoides farinae (produced by GREER) was immobilized in an immunoplate (produced by Nalge Nunc International). The dog serum obtained by immunization with the antigen extracted from Dermatophagoides farinae was reacted with the antigen. It was confirmed that GST-FcεRIα did not react with either Dermatophagoides-farinae-positive dog serum subjected to heat treatment at 56 ° C for 1 hour or with purified dog IgG, but reacted with recombinant dog IgE (produced by BETHYL). GST-FcεRIα was considered to recognize allergen specific IgE (Fig. 3). In Fig. 3, "-" indicates untreated serum at 56 ° C for 1 hour, "+" indicates serum treated at 56 ° C for 1 hour, and "cont." indicates unimmunized serum.

[Example 2] Analysis of a main mite allergen by Western blotting

Western blot analysis was performed using the sera and blood plasma samples from eight dogs. The eight dogs had developed atopic dermatitis and were diagnosed with mite allergies based on intracutaneous reactions using the solution of an antigen extracted from a Dermatophagoides farinae allergen (produced by GREER) and the ELISA method using the established recombinant dog FcεRIα chain in Example 1. β-mercaptoethanol was added to 100.0 µl of the solution of an antigen extracted from Dermatophagoides farinae to a final concentration of 50.0 µl / ml. 200 µl of the Laemmli sample buffer prepared in this way (produced by BIO-RAD) was added, followed by heat treatment at 100 ° C for 5 minutes. The resultant was applied to polyacrylamide gel (PAGEL; produced by ATTO) with a gel concentration between 5% and 20%, and then electrophoresis was performed. After electrophoresis was completed, the resultant was transferred to a PVDF membrane (Hybond-P; produced by Amersham Biosciences). The membrane was allowed to stand at 4 ° C overnight in a blocking solution (PBST (prepared by adding Tween20 to PBS to a final concentration of 0.1%) supplemented with 5% skim milk). After the membrane had been washed in PBST for 10 minutes, each dog serum or blood plasma sample was diluted 10 times in a blocking solution. Each sample in dilute solution was added to the membrane, followed by 3 hours of reaction at room temperature. Three washes (10 minutes each) were performed with PBST. Biotin labeled dog FcεRIα was diluted with a blocking solution and then the diluted solution was added to the membrane, followed by 2 hours of reaction at room temperature. After 3 washes (10 minutes each) with PBST, a streptavidin-HRP conjugate (produced by Amersham Biosciences) diluted 10,000 times with PBST was added to the membrane, followed by 1 hour of reaction at room temperature. After 5 washes (10 minutes each) with PBST, a reaction solution for a Western ECL Plus transfer detection system (produced by Amersham Biosciences) was added to the membrane, followed by 5 minutes of reaction at room temperature . The signals were then detected using X-ray film (Hyperfilm ECL; produced by Amersham Biosciences). As a result, a protein was detected that showed a strong reaction between a band corresponding to a molecular weight of 150 kDa and a band corresponding thereto of 250 kDa (Fig. 4). In Fig. 4, a band indicated with an arrow corresponds to the protein that shows a strong reaction and has a molecular weight between 150 kDa and 250 kDa. In Fig. 4, numbers 1 to 8 separately indicate eight dogs, and "ct." indicates a negative serum.

[Example 3] Analysis of an allergic mite protein by 2-D electrophoresis (bi-dimensional)

An allergenic protein with a molecular weight between 150 kDa and 250 kDa was isolated, with which IgE had reacted strongly, it was isolated by 2-D (bi-dimensional) electrophoresis. 2-D (bi-dimensional) electrophoresis was performed using a Protean IEF cell (produced by BIO-RAD). 1.0 mg of an antigen extracted from Dermatophagoides farinae (produced by GREER) was dissolved in 1.0 ml of a swelling buffer (2-D starter kit; produced by BIO-RAD). 300 µl of the solution thus obtained was swollen under active conditions (50 V, 20 ° C, and 12 hours) using 17 cm long IPG Ready strip gel (pH 4-7; produced by BIO-RAD) and a cuvette of focus. After swelling, the approach was performed under the following conditions. First, a procedure was performed to remove excess salts in step 1 (250 V, 20 minutes, and 20 ° C). In stage 2, the voltage was raised from 250 V to 10,000 V for 6 hours. In stage 3, the approach was carried out with a voltage of 10,000 V and adding the voltage hours 60,000 VH. Prior to 2-D (bi-dimensional) electrophoresis, the IPG ready strip gel was gently shaken for 10 minutes using SDS-PAGE I equilibration buffer (6 M urea, 0.375 M Tris pH 8.8, 2% SDS , 20% glycerol, and 2% DTT (w / v); produced by BIO-RAD). Subsequently, the gel was further gently stirred for 10 minutes using SDS-PAGE II equilibration buffer (6 M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, and 2.5% iodoacetamide (p / v); produced by BIO-RAD), thus balancing. The balanced IPG ready strip gel was made to adhere closely to ready PII gel (8-16%; produced by BIO-RAD) using 1% low melting agarose (v / w) (produced by BIO-RAD). The electrophoresis was performed with a constant current of 40 mA (with initial voltage of 135 V and final voltage of 400 V) for approximately 3 hours. After electrophoresis, the gel was stained using Bio-Safe (produced by BIO-RAD), so that the pattern analysis for protein stain could be done (Fig. 5).

[Example 4] Identification of an allergen spot by Western blot analysis

After 2-D (bi-dimensional) electrophoresis, the protein spot was transferred to a PVDF membrane (Hybond-P; produced by Amersham Biosciences). The membrane was allowed to stand at 4 ° C overnight in a blocking solution (PBST (prepared by adding Tween20 to PBS to a final concentration of 0.1%) supplemented with 5% skim milk). After washing the membrane with PBST for 10 minutes, a sample of dog serum or blood plasma was diluted 10 times using a blocking solution. The solution diluted in this way was added to the membrane, followed by 3 hours of reaction at room temperature. After 3 washes (10 minutes each) with PBST, biotin-labeled dog FcεRIα was diluted with a blocking solution. Diluted biotin-labeled dog FcεRIα was added to the membrane, followed by 2 hours of reaction at room temperature. After 3 washes (10 minutes each) with PBST, a streptavidin-HRP conjugate (produced by Amersham Biosciences) diluted 10,000 times with PBST was added to the membrane. One hour of reaction was carried out at room temperature. After 5 washes (10 minutes each) with PBST, a reaction solution for a Western ECL plus transfer detection system (produced by Amersham Biosciences) was added to the membrane, followed by 5 minutes of reaction at room temperature. The signals were detected using X-ray film (Hyperfilm ECL; produced by Amersham Biosciences). As a result, a spot was detected showing a strong reaction between a band corresponding to a molecular weight of 150 kDa and a band corresponding thereto of 250 kDa and at a pH of approximately 4.5. Therefore, the spot corresponding to the allergenic protein (Zen1) of the present invention was obtained (Fig. 6). In Fig. 6: the upper left Fig. 6-1 shows the 2-D (bi-dimensional) electrophoresis pattern (pH 4 to 7) of Dermatophagoides farinae; The upper right Fig. 6-2 shows the result of Western blot analysis (pH 4 to 7) using Dermatophagoides-farinae-positive serum from a patient dog that developed atopic dermatitis; Fig. 6-3 bottom left shows the 2-D (bi-dimensional) electrophoresis pattern (pH 3.9 to 5.1) of Dermatophagoides farinae; and Fig. 6-4 bottom right shows the reaction spot (where the spot appears as a black circular spot on the top in the figure and is indicated by an arrow) obtained by Western blot analysis (pH 3.9 to 5.1) using farinae-positive Dermatophagoides serum from a patient dog that developed atopic dermatitis, compared to the 2-D (bi-dimensional) electrophoresis pattern. A strong reaction was observed for the stain indicated by the arrow.

[Example 5] Proteome analysis of the Zen1 protein

The stain of the Zen protein isolated by 2-D electrophoresis (bi-dimensional) was excised from the gel and then MS / MS analysis was performed. Many MS / MS data could be obtained, but no successes were confirmed. Five peptides that were considered to be new proteins were subjected to the de novo sequencing method, so that the amino acid sequences were determined (SEQ ID NO: 3 to 7) (Table 1). A BLAST search was performed for these amino acid sequences, but clear successes were not obtained.

Table 1

Partial sequence 415: MKSLLNEANELLK Partial sequence 445: SAQDVLEK Partial sequence 847: FMQSLLNEADELLR Partial sequence 448: LPDSDLKDELAK Partial sequence 491: LPDSDLKNELAEK

Partial amino acid sequences of Zen1 determined by the de novo sequencing method.

[Example 6] Zen1 protein peptide mapping analysis

The stain of the Zen1 protein isolated by 2-D (two-dimensional) electrophoresis was excised from the gel. A peptide map was prepared and then amino acid sequencing was performed for 8 peaks. As a result, the sequences (SEQ ID NO: 8 to 18) of 11 amino acid fragments (Table 2) were determined. A BLAST search was performed, but no clear successes were obtained.

Table 2

Partial Sequence 21

: MYNFHLEAY

Partial Sequence 28

: IAHFLELE

Partial Sequence 32

: IAHFELE

Partial sequence 23-1: KFQSLLNEAN Partial sequence 23-2: IAHLESE (T) Partial sequence 24: KFQSLLN (E) A Partial sequence 22: DAQLEXE Partial sequence 9-1: SAQDVSL Partial sequence 9-2: RNEMNE Partial sequence 20-1 : MFQSLLNKADFD

Partial sequence 20-2: DLARDVXL Amino acid sequences corresponding to peaks obtained by mapping Zen 1 peptides

[Example 7] Analysis of the N-terminal amino acid sequence of the Zen1 protein

The stain of Zen1 protein isolated by 2-D electrophoresis (bi-dimensional) was excised from the gel. The N-terminal amino acid sequence (SEQ ID No. 19) of the Zen1 protein was determined by a conventional method using an HP G1005A protein sequencing system (Table 3). A BLAST search was performed for the sequence, but no clear hits were obtained.

Table 3

N-terminal sequence: DNRDDVLKQTEE

Zen1 N-terminal amino acid sequence

10 [Example 8] Extraction of total mite RNA and separation of poly (A) mite mRNA

Untreated mite bodies obtained by cultivation and growth of Dermatophagoides farinae were placed according to a conventional method in approximately 2.0 L of a saturated saline solution. The solution is

15 stirred well and then allowed to stand for 30 minutes. The mite bodies in the supernatant were purified using a strainer, washed using physiological saline, and then dried. 1.0 g of mite bodies were subjected to total RNA extraction and separation of poly (A) mite mRNA using a FastTrack 2.0 kit (produced by Invitrogen) according to the kit manual.

20 [Example 9] Synthesis of mite cDNA

Reverse transcription reaction was performed using 100 ng of the mite poly (A) mite separated in Example 8 as a template and a cDNA synthesis kit (ReverTraAce-α-; produced by Toyobo) according to the kit manual.

25 [Example 10] Zen 1 gene amplification by PCR

Based on the N-terminal amino acid sequence of the Zen1 protein, primers N-1 (5'-GAYGAYGTNTTRAARCARACNGARGAR -3 '(SEQ ID NO. 20): Y = C or T, N = A or C or G or T, and R = A or G) and N-2 (5 '-GAYGAY GTN CTN AAR CAR ACN GAR GAR -3' (SEQ ID NO. 21): Y = C oT, N = A oC or G oT, and R = A oG) 30 as sense primers. In addition, 12 primers (SEQ ID NO: 22 to 33) were designed based on the amino acid sequences obtained by the de novo sequencing method (Table 4) as reverse primers. With the use of 10 µg of the mite cDNA synthesized in Example 9 as a template, Ex taq polymerase (produced by TaKaRa Bio), and each sample prepared according to the manual, thermal denaturation treatment was performed at 94 ° C for 2 minutes and 35 reaction cycles were performed, each of which consisted of 94 ° C for 1 minute, 65 ° C for 2 minutes, and 72 ° C for 3 minutes. After performing an additional reaction at 72 ° C for 9 minutes, the sample was stored at 4 ° C. A DNA fragment of approximately 1,000 bp was obtained when PCR was performed using a 415-4 reverse primer (5 '-RTTNAGNAGRTCYTTNGCRTCYTT -3' (SEQ ID NO. 25): N = A or C or G or T, R = A or G, and Y = C or T). A DNA fragment of approximately 880 bp was obtained when PCR was performed using 491-2 (5 '-RTT RTC NGC NAG RTC YTT RTT -3' (SEQ ID NO: 29): N = A or C or G or

40 T, R = AoG, eY = CoT).

Table 4

N-1: 5 '-GAY GAY GTN TTR AAR CAR ACN GAR GAR -3' N-2: 5 '-GAY GAY GTN CTN AAR CAR ACN GAR GAR -3' 415-1: 5 '-RTT RAA RAA RTC YTT NGC RTC YTT RAA -3 '415-2: 5' -RTT NAG RAA RTC YTT NGC RTC YTT -3 '415-3: 5' -RTT RAA NAG RTC YTT NGC RTC YTT -3 '415-4: 5' - RTT NAG NAG RTC YTT NGC RTC YTT -3 '445-1: 5' -RTT RTC RAA NAC YTC RTG NGC -3 '445-2: 5' -RTT RTC NAG NAC YTC RTG NGC -3 '491-1: 5 '-RTT RTC NGC RAA RTC YTT RTT -3' 491-2: 5 '-RTT RTC NGC NAG RTC YTT RTT -3' 448-1: 5 '-RTT NGC RAA RTC YTC RTT RAA YTC -3' 448-2 : 5 '-RTT NGC NAG RTC YTC RTT RAA YTC -3' 448-3: 5 '-RTT NGC RAA RTC YTC RTT NAG YTC -3' 448-4: 5 '-RTT NGC NAG RTC YTC RTT NAG YTC -3 '

Mixing of primer sequences synthesized for amplification of the Zen 1 gene. A fragment of approximately 1000 bp was obtained when PCR was performed using N-1 and 415-4. A fragment of approximately 880 bp was obtained when PCR was performed

using N-1 and 491-2. N = A or C or G or T, R = A or G, Y = C or T

[Example 11] Cloning of the Zen 1 gene

The DNA fragment amplified using primers N-1 and 415-4 in Example 10 was collected from agarose gel (SUPREC-O1 produced by TaKaRa). The DNA fragment was ligated to the cloning site of the pGEM-T Easy vector (produced by Promega) using T4 DNA ligase, thereby transforming the Escherichia coli TOP10 host (produced by Invitrogen). Specifically, competent Escherichia coli cells and a plasmid were mixed and then the mixture was subjected to temperature treatment on ice for 30 minutes, at 42 ° C for 30 seconds, and on ice for 2 minutes. The resultant was then suspended in an SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose), followed by 1 hour of incubation at 37 ° C. Subsequently, the transformed Escherichia coli were grown at 37 ° C overnight in an LB agar medium (1% yeast extract, 0.5% tryptone, and 1% NaCl) supplemented with 50 µg / ml ampicillin, thus obtaining colonies of Escherichia coli. The white clones that were thought to contain the inserted fragment were selected. The clones were grown overnight in an LB medium supplemented with 50 µg / ml ampicillin. Plasmid DNA was purified using a GFX (registered trademark) Micro Plasmid Prep kit (produced by Amersham Bioscience). The sequencing reaction was performed using a cyclic sequencing kit with dye primer (produced by Amersham) and then nucleotide sequence analysis was performed using a fluorescence DNA sequencer (produced by Shimadzu Corporation). In addition, a final determination was made when the nucleotide sequences of 3 clones were found to coincide completely after nucleotide sequence analysis thereof (Fig. 7-1 and Fig. 7-2).

[Example 12] Zen1 gene analysis

The Zen1 gene cloned in Example 11 was analyzed using Genetyx-win software ver.6 (produced by Software Development). The amount of bases was 1020 bp and the amount of amino acid residues was 340 (Fig. 7-1, Fig. 7-2, and SEQ ID NO: 1 and 2). A BLAST search was performed for the nucleotide sequence and amino acid sequence of the gene, but no clear hits were obtained. The Zen1 gene was thought to be a new gene.

[Example 13] Isolation of the full length Zen1 cDNA

Full length cDNA was isolated by the RACE method (rapid amplification of cDNA ends). Total RNA was extracted from the mite bodies used in Example 8 using an SV total RNA isolation kit (produced by Promega). A mold for RACE was prepared using a GeneRacer kit (registered trademark) (produced by Invitrogen) according to the kit manual. In addition, based on the partial sequences of Zen1 obtained in Example 12, primers were synthesized for the 1st PCR and the nested PCR for amplification of the 5 'and 3' ends (Table 5). The amplification reaction for the 5 'and 3' ends was performed as follows. To 1.0 µl of the RACE mold prepared above, 3.0 µl of each of the 5 'and 3' primers included in a GeneRacer kit (registered trademark), 1.0 µl of a dNTP mixing solution was added (10 mM each), 5.0 µl of 10 x cDNA PCR reaction buffer included in the Advantage cDNA polymerase mixture (produced by CLONTECH), 1.0 µl Advantage cDNA polymerase mixture, and 1 , 0 µl of each of the gene specific primers for the 1st PCR synthesized above and adjusted to 10 µM. The volume of the resulting solution was then adjusted to 50.0 µl using sterile distilled water. Gene amplification was performed using a Touchdown PCR method. The prepared sample solution was subjected to thermal denaturation at 94 ° C for 1 minute, 5 reaction cycles, each consisting of 94 ° C for 30 seconds and 72 ° C for 4 minutes, 5 reaction cycles, each of which it consisted of 94 ° C for 30 seconds and 70 ° C for 4 minutes, 25 cycles of final reaction, each of which consisted of 94 ° C for 30 seconds and 68 ° C for 4 minutes, and then storage at 4 ° C. After completion of the 1st PCR, at 1.0 µl of each of the 5 'and 3' end amplification reaction solutions, 1.0 µl of each 5 'primer and 3' primer were added for the nested PCR included in a GeneRacer kit (registered trademark), 1.0 µl of a dNTP mixing solution (10 mM each), 5.0 µl of 10 x cDNA PCR reaction buffer included in the Advantage cDNA polymerase mixture (produced by CLONTECH), 1.0 µl of Advantage cDNA polymerase mixture, and 1.0 µl of each of the gene-specific primers for the 1st PCR synthesized above and adjusted to 10 µM. The volume of the resulting solution was then adjusted to 50.0 µl using sterile distilled water. Gene amplification was performed using the previous Touchdown PCR method. The amplified fragments thus obtained from the 5 'and 3' ends were confirmed by electrophoresis using 1.0% agarose gel. These fragments were cleaved and then collected (SUPREC-01 produced by TaKaRa). The fragments were ligated to the cloning site of the pGEM-T Easy vector (produced by Promega) using T4 DNA ligase, thereby transforming the Escherichia coli TOP10 host (produced by Invitrogen). Specifically, competent Escherichia coli cells and a plasmid were mixed and then the mixture was subjected to temperature treatment on ice for 30 minutes, at 42 ° C for 30 seconds, and on ice for 2 minutes. The resultant was then suspended in an SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM Glucose), followed by 1 hour of incubation at 37 ° C. Subsequently, the transformed Escherichia coli were grown at 37 ° C overnight in an LB agar medium (1% yeast extract, 0.5% tryptone, and 1% NaCl)

supplemented with 50 µg / nl ampicillin, thereby obtaining colonies of Escherichia coli. The white colonies that were thought to contain the inserted fragments were selected. The clones were grown overnight in an LB medium supplemented with 50 µg / ml ampicillin. Plasmid DNA was purified using a GFX (registered trademark) Micro Plasmid Prep kit (produced by Amersham Bioscience). The sequencing reaction was performed using a cyclic sequencing kit with dye primer (produced by Amersham) and then nucleotide sequence analysis was performed using a fluorescence DNA sequencer (produced by Shimadzu Corporation). In addition, a final determination was made when it was found that the nucleotide sequences of 3 clones completely coincided after their analysis. As a result, the nucleotide sequence of the 5 'end and that of the 3' end of Zen1 could be determined (Fig. 8-1 to Fig. 8-4 and SEQ ID NO: 34 and 35).

Table 5

Primer Name

Sequence Purpose of use

Zen1 RS-1:

5 '-AAT TAC AAA CAT GAG TTA GAA -3' 3 'RACE 1st PCR

Zen1 RS-2:

5 '-GAA TTG TTG ACA ATG TTC AAA -3' 3 'RACE nested PCR

Zen1 RR-1:

5 '-GAT TTC ATC TTT CAA ATC TGA -3' 5 'RACE 1st PCR

Zen1 RR-2:

5 '-CTT TTC CAA TAC ATC CTG GGC -3' 5 'RACE nested PCR

(From above, SEQ ID No. 36, 37, 38, and 39) Primers used in the RACE method and the sequences thereof

[Example 14] Purification of recombinant Zen1

The Zen1 cDNA obtained in Example 13 to which sites for restriction enzymes BamHI and XhoI had been added was amplified by PCR. The amplification product was ligated to the BamHI and XhoI sites of the Escherichia coli pGEX4T-1 expression plasmid vector (produced by Amersham Biosciences) using T4 DNA ligase. The strain of

E. coli TOP10 (produced by Invitrogen) was transformed with the recombinant plasmid thus obtained. The transformed strain was grown at 37 ° C overnight in an LB medium containing 100 µg / ml ampicillin. A small amount of the strain was subculitted in a new LB medium until the OD at 600 nm reached 1.0. Next, IPTG (isopropyl-1-thio-β-D-galactoside) was added to a final concentration of 1 mM. Three hours later, the cells were collected and then washed once with PBS (pH 7.4). The cells collected again were lysed by ultrasonication in PBS (pH 7.4), a fraction was removed by centrifugation, and then a soluble fraction containing Zen1 fused to glutathione S-transferase (GST) was collected. Subsequently, Zen1 fused to GST was obtained from the soluble fraction using a glutathione sepharose 4B column (produced by Amersham Biosciences). Thrombin (produced by Amersham Biosciences) was added in an amount 1/100 of that of the fusion protein to a solution containing a purified fusion product (i.e., Zen1 fused to GST), followed by 20 hours of reaction at 22 ºC. Therefore, GST was separated from Zen1. Subsequently, thrombin was removed using Benzamidine Sepharose (produced by Amersham Biosciences) and then a recombinant Zen1 was purified. The purified Zen1 obtained in this way showed a single band corresponding to a molecular weight of approximately 60 kDa confirmed by SDS-PAGE (Fig. 9).

[Example 15] Preparation of a polyclonal anti-Zen1 antibody and analysis of the reactivity of the antibody with the mite protein

Six mice (BALB / c, female, 4 weeks old) were immunized 5 times with the purified recombinant Zen1 in Example 14 at 1 week intervals. Subsequently, blood was collected from the mice, the sera were separated, and then the reaction of the IgG antibody was analyzed by ELISA. The ELISA was performed as follows. 1.0 µg of the recombinant Zen1 and 4.0 µg of an antigen extracted from Dermatophagoides farinae (produced by GREER) were immobilized separately in immunoplates (produced by Nalge Nunc International), followed by 1 hour of blocking at 37 ° C using a blocking solution (prepared by adding Tween20 to PBS supplemented with 10% FBS to a final concentration of 0.05%). Each mouse serum sample was diluted 1000 times with a blocking solution and then allowed to react at room temperature for 1 hour. After washing each immunoplate, a goat monoclonal antibody labeled with HRP anti-mouse IgG (produced by ZYMED) was diluted 2000 times with a blocking solution therewith. After washing each immunoplate, 100 µl of an enzyme substrate solution (ABTS solution) was added to each well, followed by 10 minutes of reaction at 37 ° C. The enzymatic reaction was stopped by adding 100 µl of a 0.32% sodium fluoride solution to each well. The absorbance of each well at 414 nm was measured using an immunolector (BioRad). After confirming the IgG reaction with the relevant subject by ELISA, it was determined that the subject was a polyclonal antibody against Zen1.

The reactivity of mouse IgG with the polyclonal antibody (the recombinant Zen1) and the same with respect to mite bodies by Western blotting was analyzed. 200 µl of a Laemmli sample buffer (produced by BIO-RAD) was prepared by adding β-mercaptoethanol to a final concentration of 50.0 µl / ml to 100.0 µl of the recombinant Zen1 protein solution adjusted to 50 µg / ml and 100.0 µl of the solution of an antigen extracted from Dermatophagoides farinae. After heat treatment at 100 ° C for 5 minutes, the resultant was applied to polyacrylamide gel (PAGEL; produced by ATTO) with a gel concentration between 5% and 20%. Therefore, electrophoresis was performed. After the electrophoresis was completed, the resultant was transferred to a membrane of

PVDF (Hybond-P; produced by Amersham Biosciences). The membrane was allowed to stand at 4 ° C overnight in a blocking solution (PBST (prepared by adding Tween20 to PBS to a final concentration of 0.1%) supplemented with 5% skim milk). The membrane was washed with PBST for 10 minutes. A goat monoclonal antibody labeled with HRP anti-mouse IgG (produced by ZYMED) was diluted 20,000 times with 5 PBST, and then the diluted solution was added to the membrane, followed by 2 hours of reaction at room temperature. After 5 washes (10 minutes each) with PBST, the reaction solution of a Western ECL Plus transfer detection system (produced by Amersham Biosciences) was added to the membrane, followed by 5 minutes of reaction at room temperature. The signals were detected using X-ray film (Hyperfilm ECL; produced by Amersham Biosciences). As a result, a signal indicating the reaction with

Recombinant Zen1 with a molecular weight of approximately 60 kDa and a signal indicating the reaction with wild-type Zen1 with a molecular weight between 150 kDa and 250 kDa (Fig. 10). Accordingly, it was confirmed that the full length Zen1 cDNA isolated in Example 13 encodes said mite allergen protein with a molecular weight between 150 kDa and 250 kDa.

[Example 16] Analysis of IgE reactivity of recombinant Zen1

The allergenicity of recombinant Zen1 purified in Example 14 was evaluated by ELISA. 1.0 µg of recombinant Zen1 was immobilized in an immunoplate (produced by Nalge Nunc International KK), followed by blocking at 37 ° C for 1 hour with a blocking solution (prepared by adding Tween20 to PBS supplemented with 10% FBS 20 to a final concentration of 0.05%). The sera of nine dogs that tested positive for Dermatophagoides farinae (sera of dogs that tested positive for Dermatophagoides farinae confirmed by intracutaneous reaction were reacted using an antigenic solution of Dermatophagoides farinae (produced by GREER) diluted 50 times using saline solution) physiological)) with a negative control dog serum. Biotin-labeled GST-FcεRIα was added and then a color development reaction was caused by the addition of peroxidase-conjugated streptavidin (produced by Jackson Immuno Research) and an enzyme substrate solution (ABTS solution). The enzymatic reaction was stopped by the addition of 100 µl of a 0.32% sodium fluoride solution per well. The absorbance of each well at 414 nm was measured using an immunolector (BioRad). Specifically, the serum IgE reaction was analyzed (from nine dogs that had tested positive for mites confirmed by intracutaneous reaction of this procedure and the

30 negative dog (control)) to recombinant Zen1 by an ELISA system using recombinant dog FcεRIα. As a result, values above the value for the negative dog (indicated with a dashed line) were confirmed. Therefore, it was confirmed that recombinant Zen1 is an allergenic protein that reacts with IgE (Fig. 11).

Industrial applicability

The present invention makes it possible to provide safe and effective recombinant mite allergens as therapeutic agents or diagnostic agents for allergic diseases to mites, which do not contain impurities that induce anaphylaxis.

40 SEQUENCE LIST

<110> NIPPON ZENYAKU KOGY0 LTD.

<120> New mite allergen 45

<130> PH-2409-PCT

<150> JP2004-116089

<151> 50

<160> 39

<170> PatentIn Ver. 2. 1

55 <210> 1

<211> 1022

<212> DNA

<213> Dermatophagoides farinae

60 <220>

<221> CDS

<222> (1) .. (1020)

<400> 1 65

<210> 2

<211> 340

<212> PRT

<213> Dermatophagoides farinae

<400> 2

<210> 3

<211> 13 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 10

<400> 3

15 <210> 4

<211> 8

<212> PRT

<213> Artificial sequence

20 <220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 4

<210> 5

<211> 14

<212> PRT 30 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide

35 <400> 5

<210> 6

<211> 12 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 10

<400> 6

15 <210> 7

<211> 13

<212> PRT

<213> Artificial sequence

20 <220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 7

<210> 8

<211> 9

<212> PRT 30 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide

35 <400> 8

<210> 9 40 <211> 8

<212> PRT

<213> Artificial sequence

<220> 45 <223> Description of the artificial sequence: Synthetic peptide

<400> 9

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence 5

<220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 10 10

<210> 11

<211> 10 15 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 20

<400> 11

25 <210> 12

<211> 8

<212> PRT

<213> Artificial sequence

30 <220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 12

<210> 13

<211> 9

<212> PRT 40 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide

45 <400> 13

<210> 14 50 <211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 14

<210> 15

<211> 7 10 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 15

<400> 15

20 <210> 16

<211> 6

<212> PRT

<213> Artificial sequence

25 <220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 16

<210> 17

<211> 12

<212> PRT 35 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide

40 <400> 17

<210> 18 45 <211> 8

<212> PRT

<213> Artificial sequence

<220> 50 <223> Description of the artificial sequence: Synthetic peptide

<400> 18

<210> 19

<211> 12 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 10

<400> 19

15 <210> 20

<211> 27

<212> DNA

<213> Artificial sequence

20 <220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base 25 <222> 9y21

<223> n represents a, c, g or t

<400> 20

Gaygaygtnt Traarcarac Ngargar 27 30

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence 35

<220>

<223> Description of the artificial sequence: primer

<220> 40

<221> modified base

<222> 9, 12 and 21

<223> n represents a, c, g or t 45

<400> 21 gaygaygtnc tnaarcarac ngargar 27

<210> 22 50 <211> 27

<212> DNA

<213> Artificial sequence

<220> 55 <223> Description of the artificial sequence: primer

<220>

<221> modified base

60 <222> 16

<223> n represents a, c, g or t

<400> 22 rttraaraar tcyttngcrt cyttraa 27 5

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220> 15 <221> modified base

<222> 4 and 16

<223> n represents a, c, g or t

<400> 23 rttnagraar tcyttngcrt cytt 24

<210> 24

<211> 24

<212> DNA 25 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base

<222> 7 and 16

<223> n represents a, c, g or t

35 <400> 24 rttraanagr tcyttngcrt cytt 24

<210> 25

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer 45

<220>

<221> modified base

<222> 4, 7 and 16

<223> n represents a, c, g or t

<400> 25 rttnagnagr tcyttngcrt cytt 24

55 <210> 26

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base 65 <222> 10 and 19

<223> n represents a, c, g or t

<400> 26 rttrtcraan acytcrtgng c 21

<210> 27 5 <211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base

<222> 7, 10 and 19 15 <223> n represents a, c, g or t

<400> 27 rttrtcnagn acytcrtgng c 21

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

25 <220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base

<222> 7

<223> n represents a, c, g or t

<400> 28

rttrtcngcr aartcyttrt t 21 35

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220> 45 <221> modified base

<222> 7 and 10

<223> n represents a, c, g or t

<400> 29 rttrtcngcn agrtcyttrt t 21

<210> 30

<211> 24

<212> DNA 55 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

<220>

<221> modified base

<222> 4

<223> n represents a, c, g or t

65 <400> 30 rttngcraar tcytcrttra aytc 24

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence 5

<220>

<223> Description of the artificial sequence: primer

<220> 10 <221> modified base

<222> 4 and 7

<223> n represents a, c, g or t

<400> 31 15 rttngcnagr tcytcrttra aytc 24

<210> 32

<211> 24

<212> DNA 20 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer

25 <220>

<221> modified base

<222> 4 and 19

<223> n represents a, c, g or t

30 <400> 32 rttngcraar tcytcrttna gytc 24

<210> 33

<211> 24 35 <212> DNA

<213> Artificial sequence

<220>

<223>

Description of the artificial sequence: primer 40

<220>

<221> modified base

<222> 4, 7 and 19

<223>

n represents a, c, g or t 45

<400> 33 rttngcnagr tcytcrttna gytc 24

<210> 34 50 <211> 1518

<212> DNA

<213> Dermatophagoides farinae

<220> 55 <221> CDS

<222> (1) .. (1515)

<400> 34

<210> 35

<211> 505

<212> PRT

<213> Dermatophagoides farinae

<400> 35

<210> 36

<211> 21 5 <212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: primer 10

<400> 36 aattacaaac atgagttaga a 21

<210> 37 15 <211> 21

<212> DNA

<213> Artificial sequence

<220> 20 <223> Description of the artificial sequence: primer

<400> 37 gaattgttga caatgttcaa a21

25 <210> 38

<211> 21

<212> DNA

<213> Artificial sequence

30 <220>

<223> Description of the artificial sequence: primer

<400> 38

gatttcatct ttcaaatctg a 21 35

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence 40

<220>

<223> Description of the artificial sequence: primer

<400> 39 45 cttttccaat acatcctggg c 21

Claims (14)

1. The following recombinant mite allergen (a) to (c) that has mite allergenic activity: 5 (a) a recombinant mite allergen comprising the amino acid sequence represented by SEQ ID NO: 35; (b) a recombinant mite allergen comprising the amino acid sequence of SEQ ID No. 35, except that 1 to 10 amino acids have been deleted from the amino acid sequence represented by SEQ ID No. 35; or 10 (c) a recombinant mite allergen comprising an amino acid sequence that has at least 85% sequence homology with the amino acid sequence of SEQ ID No. 35 calculated using BLAST and using default parameters. 2. A gene that (1) encodes the following mite allergen (a) to (c) that has mite allergenic activity: 15

(to)

a mite allergen comprising the amino acid sequence represented by SEQ ID NO: 35;

(b)

an mite allergen comprising the amino acid sequence of SEQ ID No. 35, except that 1 to 10 amino acids have been deleted from the amino acid sequence represented by SEQ ID No. 35; or

(C)

a recombinant mite allergen comprising an amino acid sequence that has at least 85

20% sequence homology with the amino acid sequence of SEQ ID No. 35 calculated using BLAST and using default parameters; or

(2)

It comprises the following DNA (d) or (e):

(d)

a DNA comprising the nucleotide sequence represented by SEQ ID NO: 34; or

(and)

a DNA that encodes a protein that has mite allergenic activity and that hybridizes under conditions

Rigorous with a DNA comprising a sequence complementary to that of the DNA comprising the nucleotide sequence of SEQ ID No. 34, wherein the DNA (1) hybridizes in the presence of 0.7 M NaCl at 1.0 M at 68 ºC using a filter on which DNA has been immobilized and washing with the use of SSC solution 0.1 at 2 x 68 ºC, or (2) It may form a hybrid when it is transferred to and immobilized on a nitrocellulose membrane by the Southern transfer method and then allowed to react overnight at 42 ° C in a buffer. 30 hybridization of 50% formamide, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's solution and 100 µg / ml salmon sperm DNA. 3. The mite allergen according to claim 1 part (c) or the gene according to claim 2 part (c), where the homology level is at least 90%. 35 4. The mite allergen or gene according to claim 3, wherein the homology level is at least 95%.

5.

The mite allergen or gene according to claim 3, wherein the homology level is at least 40 97%.

6. A recombinant vector, which contains the gene according to any one of claims 2 to 5.

7.

A fusion protein, which is composed of the mite allergen according to any one of claims 1 and 3 to 5 and another protein.

8. A bacterial, yeast, insect or animal cell, which is transformed using the recombinant vector according to claim 6. 9. A method of producing a recombinant mite allergen, comprising culturing the bacterial, yeast, insect or animal cell according to claim 8 under conditions where the gene can express itself, causing the cell to produce a recombinant allergen. of mite, and then collect the recombinant mite allergen. A method of producing a recombinant mite allergen, which comprises culturing a bacterial, yeast, insect or animal cell that is transformed with a recombinant vector containing a gene that encodes a fusion protein between the mite allergen of any one of claims 2-5 and another protein under conditions in which the gene can be expressed, cause the cell to produce a recombinant fusion mite allergen, collect the recombinant fusion mite allergen and then remove the other protein fused to the 60 allergen 11. A therapeutic agent for mite allergic diseases, which contains as an active principle the recombinant mite allergen according to any one of claims 1 and 3 to 5, or the fusion protein according to claim 7. 12. A diagnostic agent for mite allergic diseases, which contains as an active principle the recombinant mite allergen according to any one of claims 1 and 3 to 5, or the fusion protein according to claim 7. An antibody against the mite allergen according to any one of claims 1 and 3 to 5. 14. The antibody against the mite allergen according to claim 13, which is a monoclonal antibody.

fifteen.

A hybridoma, which produces the monoclonal antibody according to claim 14. 10

16. An immunoassay method for a house dust mite allergen, which uses the antibody according to any one of claims 13 to 15.

17.

The immunoassay method for a house dust mite allergen according to claim 16, 15 which is the ELISA method.